Jammallo Lauren, Eidell Keith, Davis Paul H, Dufort Fay J, Cronin Courtney, Thirugnanam Sivasakthivel, Chiles Thomas C, Roos David S, Gubbels Marc-Jan
Department of Biology, Boston College, Chestnut Hill, MA, USA.
Mol Biochem Parasitol. 2011 Feb;175(2):133-43. doi: 10.1016/j.molbiopara.2010.10.007. Epub 2010 Oct 28.
Toxoplasmosis is characterized by fast lytic replication cycles leading to severe tissue lesions. Successful host cell invasion is essential for pathogenesis. The division cycle of Toxoplasma gondii is characterized by an unusual cell cycle progression and a distinct internal budding mechanism. To identify essential genes involved in the lytic cycle we devised an insertional gene trapping strategy using the Tet-transactivator system. In essence, a random, active promoter is displaced with a tetracycline regulatable promoter, which if in an essential gene, will result in a conditionally lethal phenotype upon tetracycline addition. We isolated eight mutants with growth defects, two of which displayed modest invasion defects, one of which had an additional cell cycle defect. The trapped loci were identified using expression microarrays, exploiting the tetracycline dependent expression of the trapped genes. In mutant 3.3H6 we identified TCP-1, a component of the chaperonin protein folding machinery under the control of the Tet promoter. However, this gene was not critical for growth of mutant 3.3H6. Subsequently, we identified a suppressor gene encoding a protein with a hypothetical function by guided cosmid complementation. In mutant 4.3B13, we identified TAF250, an RNA polymerase II complex component, as the trapped, essential gene. Furthermore, by mapping the plasmid insertion boundaries we identified multiple genomic rearrangements, which hint at a potential replication dependent DNA repair mechanism. Furthermore, these rearrangements provide an explanation for inconsistent locus rescue results observed by molecular biological approaches. Taken together, we have added an approach to identify and study essential genes in Toxoplasma.
弓形虫病的特征是快速的裂解复制周期，可导致严重的组织损伤。成功的宿主细胞入侵对于发病机制至关重要。刚地弓形虫的分裂周期具有不寻常的细胞周期进程和独特的内出芽机制。为了鉴定参与裂解周期的必需基因，我们设计了一种使用Tet反式激活系统的插入基因捕获策略。本质上，一个随机的活性启动子被一个四环素可调节启动子取代，如果该启动子位于一个必需基因中，添加四环素后将导致条件致死表型。我们分离出八个具有生长缺陷的突变体，其中两个表现出适度的入侵缺陷，一个具有额外的细胞周期缺陷。利用捕获基因的四环素依赖性表达，通过表达微阵列鉴定捕获的基因座。在突变体3.3H6中，我们在Tet启动子的控制下鉴定出TCP-1，它是伴侣蛋白折叠机制的一个组成部分。然而，该基因对突变体3.3H6的生长并不关键。随后，我们通过引导黏粒互补鉴定出一个编码具有假设功能蛋白质的抑制基因。在突变体4.3B13中，我们鉴定出TAF250，它是RNA聚合酶II复合物的一个组成部分，是捕获的必需基因。此外，通过绘制质粒插入边界，我们鉴定出多个基因组重排，这暗示了一种潜在的依赖复制的DNA修复机制。此外，这些重排为分子生物学方法观察到的不一致的基因座拯救结果提供了解释。总之，我们增加了一种鉴定和研究弓形虫必需基因的方法。
